There are numerous situations where one is interested in performing a multiplicity of determinations in a single vessel and being able to individually determine the result of each determination. In order to be able to achieve the individual results, it is necessary that each determination be independent of the other determinations, and that each determination provide a product that can be measured and distinguished from the products of the other determinations.
One of the areas of interest is the effect of a change in an environment on a plurality of enzymes. In screening compounds for biological activity, one is interested in the effect of the compound on one or more targets, as well as the effect of the compound on enzymes that are not targets. Therefore, if one can perform a single determination under the same conditions, so as to determine the effect of the compound on a plurality of enzymes, one can not only determine the biological activity of the compound as to targets of interest, but also the specificity of the compound in relation to side effects.
Besides screening compounds for biological activity, there is also an interest in determining the effect of a change in environment on cellular activity, as to specific enzymes. For cancer cells, one would be interested in determining changes in the cellular expression of proteins, the activity of individual enzymes, or an enzyme profile in relation to a compound or course of treatment.
One special class of enzymes for which enzyme multiplexing would be advantageous is cell-surface or intracellular receptors, which represent a significant class of targets for drug screening because the receptors are involved in cell growth and metabolism. Many receptors have enzyme domains, such as the insulin receptor, insulin-like growth factor receptor, epidermal growth factor, and platelet-derived growth factor (White). Some receptors can be coupled to enzyme for assaying the receptors (Bunemann). When a ligand binds to the specific site of a receptor, it typically activates the receptor's enzyme domain. In many cases, the enzyme is protein kinase that can be measured by determination of the rate of phosphorylation of a synthetic peptide. Since many receptors have high affinity to their specific ligands (Pike, 1984, Blakesley, Sasaki), it is possible to develop a method to perform the receptor assay by monitoring their enzyme (kinase) activity. Since only the specifically bound, natural ligand has the potential to activate the enzyme domain, the receptor assay by monitoring its enzyme activity may avoid the non-specific binding issue. This approach can be used for both ligand binding assay and enzyme assay for many receptors, especially for hormones and growth factor receptors.
In screening for compounds that are capable of inhibiting ligand/receptor binding, there are two, and sometimes three, variables that must be screened. The first variable is the test compound itself, e.g., a large number of test compounds derived from a library of potential inhibitors of ligand/receptor interaction. The second variable is test-compound concentration, relative to the concentration of receptor ligand. The third variable is the effect of the test compound on other enzymes, e.g., other receptor kinases on or in the target cell. Testing for and optimizing the concentration of test compounds, and assaying their effect on other cellular enzymes ultimately requires a large number of assay samples. By combining one or more of the test variables in a multiplexed assay, one could substantially reduce the number of assay that needed to be performed and/or provide an internal control for multiple assays.